Neuronal Inactivity Co-opts LTP Machinery to Drive Potassium Channel Splicing and Homeostatic Spike Widening.

Homeostasis of neural firing properties is important in stabilizing neuronal circuitry, but how such plasticity might depend on alternative splicing is not known. Here we report that chronic inactivity homeostatically increases action potential duration by changing alternative splicing of BK channels; this requires nuclear export of the splicing factor Nova-2. Inactivity and Nova-2 relocation were connected by a novel synapto-nuclear signaling pathway that surprisingly invoked mechanisms akin to Hebbian plasticity: Ca2+-permeable AMPA receptor upregulation, L-type Ca2+ channel activation, enhanced spine Ca2+ transients, nuclear translocation of a CaM shuttle, and nuclear CaMKIV activation. These findings not only uncover commonalities between homeostatic and Hebbian plasticity but also connect homeostatic regulation of synaptic transmission and neuronal excitability. The signaling cascade provides a full-loop mechanism for a classic autoregulatory feedback loop proposed ∼25 years ago. Each element of the loop has been implicated previously in neuropsychiatric disease.

Analysis of the relationship between lens morphology and aberrations in patients with myopia: a cross-sectional study.

PURPOSE: To investigate the relationship between lens morphology and aberrations in patients with myopia. METHODS: This cross-sectional study included 155 patients with myopia in their right eyes. Spherical power and cylindrical power were achieved by cycloplegic autorefraction. The eyes were divided into three groups for analysis based on their spherical equivalent (SE) values. The 4 mm and 6 mm ocular and internal aberrations were measured using the OPD-scan III. Lens parameters were measured using CASIA2, including lens thickness (LT), radius of anterior/posterior lens surface curvature (RAL/RPL), lens decentration (DEC), and lens tilt (TILT). The differences of lenticular parameters and aberration parameters among the three groups analyzed with ANOVA or Kruskal Wallis test. Pearson correlation or Spearman correlation analysis was performed to evaluate the relationships between the lens parameters and aberrations. A p value < 0.05 indicated statistical significance. RESULTS: The difference in LT, RAL, DEC and TITL among the three groups was statistically significant (p < 0.05). And there were differences among differences in internal high-order aberrations, spherical aberration, and coma aberration(p < 0.05).Spherical power was positively correlated with LT and TITL (p < 0.05) and negatively correlated with DEC, RAL, and RPL (p < 0.05). Cylindrical power was positively correlated with LT (p < 0.05) and negatively correlated DEC (p < 0.05); The lenticular parameters (LT, RAL, DEC, and TILT) were mainly correlated with the ocular and internal spherical aberration. LT and DEC were correlated with ocular and internal higher-order aberrations and coma aberration. CONCLUSION: DEC and LT were the main factors affecting aberrations in patients with myopia.

Lipophagy mediated carbohydrate-induced changes of lipid metabolism via oxidative stress, endoplasmic reticulum (ER) stress and ChREBP/PPARγ pathways.

High-carbohydrate diets (HCD) can induce the occurrence of nonalcoholic fatty liver disease (NAFLD), characterized by dramatic accumulation of hepatic lipid droplets (LDs). However, the potential molecular mechanisms are still largely unknown. In this study, we investigated the role of autophagy in the process of HCD-induced changes of hepatic lipid metabolism, and to examine the process of underlying mechanisms during these molecular contexts. We found that HCD significantly increased hepatic lipid accumulation and activated autophagy. Using primary hepatocytes, we found that HG increased lipid accumulation and stimulated the release of NEFA by autophagy-mediated lipophagy, and that lipophagy significantly alleviated high glucose (HG)-induced lipid accumulation. Oxidative and endoplasmic reticulum (ER) stress pathways played crucial regulatory roles in HG-induced lipophagy activation and HG-induced changes of lipid metabolism. Further investigation found that HG-activated lipophagy and HG-induced changes of lipid metabolism were via enhancing carbohydrate response element-binding protein (ChREBP) DNA binding capacity at PPARγ promoter region, which in turn induced transcriptional activation of the key genes related to lipogenesis and autophagy. The present study, for the first time, revealed the novel mechanism for lipophagy mediating HCD-induced changes of lipid metabolism by oxidative stress and ER stress, and ChREBP/PPARγ pathways. Our study provided innovative evidence for the direct relationship between carbohydrate and lipid metabolism via ChREBP/PPARγ pathway.

Zinc reduces hepatic lipid deposition and activates lipophagy via Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways.

Zinc (Zn) deficiency is the most consistently discovered nutritional manifestations of fatty liver disease. Although Zn is known to stimulate hepatic lipid oxidation, little is known about its underlying mechanism of action in lipolysis. Given the potential role of lipophagy in lipid metabolism, the purpose of this study was to test the hypothesis that Zn attenuates hepatic lipid accumulation by modulating lipophagy. The present study indicated that Zn is a potent promoter of lipophagy. Zn administration significantly alleviated hepatocellular lipid accumulation and increased the release of free fatty acids in association with enhanced fatty acid oxidation and inhibited lipogenesis, which was accompanied by activation of autophagy. Moreover, Zn reduced lipid accumulation and stimulated lipolysis by autophagy-mediated lipophagy. Zn-induced up-regulation of autophagy and lipid depletion is free Zn2+-dependent in the cytosols. Zn-induced autophagy and lipid turnover involved up-regulation of the calcium/calmodulin-dependent protein kinase kinase-ß (Ca2+/CaMKKß)/AMPK pathway. Meanwhile, Zn2+-activated autophagy and lipid depletion were via enhancing metal response element-binding transcription factor (MTF)-1 DNA binding at PPARα promoter region, which in turn induced transcriptional activation of the key genes related to autophagy and lipolysis. Zn activated the pathways of Zn2+/MTF-1/ Peroxisome proliferator-activated receptor (PPAR)α and Ca2+/CaMKKß/AMPK, resulting in the up-regulation of lipophagy and accordingly reduced hepatic lipid accumulation. Our study, for the first time, provided innovative evidence of the direct relationship between metal elements (Zn) and lipid metabolism. The present study also indicated the novel mechanism for Zn-induced lipolysis by the activation of Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways, which induced the occurrence of lipophagy. These results provide new insight into Zn nutrition and its potential beneficial effects on the prevention of fatty liver disease in vertebrates.-Wei, C.-C., Luo, Z., Hogstrand, C., Xu, Y.-H., Wu, L.-X., Chen, G.-H., Pan, Y.-X., Song, Y.-F. Zinc reduces hepatic lipid deposition and activates lipophagy via Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways.

Zn Stimulates the Phospholipids Biosynthesis via the Pathways of Oxidative and Endoplasmic Reticulum Stress in the Intestine of Freshwater Teleost Yellow Catfish.

The hypothesis of our study was that waterborne Zn exposure evoked phospholipids (PL) biosynthesis to compensate for the loss of membrane integrity, and the pathways of oxidative stress and endoplasmic reticulum (ER) stress mediated the Zn-evoked changes of PL biosynthesis. Thus, we conducted RNA sequencing to analyze the differences in the intestinal transcriptomes between the control and Zn-treated P. fulvidraco. The 56-day Zn exposure increased the intestinal Zn accumulation, and mRNA levels of 816 genes were markedly up-regulated, while that of 263 genes were down-regulated. Many differentially expressed genes in the pathways of PL biosynthesis and protein processing in ER were identified. Their expression profiles indicated that waterborne Zn exposure injured protein metabolism, induced PL biosynthesis caused oxidative stress and ER stress, and activated the unfolded protein response. Then, using the primary enterocytes, we identified the mechanism of oxidative and ER stress mediating Zn-induced PL biosynthesis, and indicated that the activation of these pathways constituted adaptive mechanisms to reduce Zn toxicity. Our study demonstrated that Zn exposure via the water increased Zn accumulation and PL biosynthesis, and that oxidative stress and ER stress were interdependent and mediated the Zn-induced PL biosynthesis of the intestine in the freshwater teleost.

Six indicator genes for zinc (Zn) homeostasis in freshwater teleost yellow catfish Pelteobagrus fulvidraco: molecular characterization, mRNA tissue expression and transcriptional changes to Zn exposure.

Excessive Zn in the aquatic environment can be toxic and causes dysfunction in Zn homeostasis for fish, which ultimately influences the function of various biological processes. Zn homeostasis is controlled by Zn transporters. This study cloned and characterized the full-length cDNA sequences of six Zn transport-relevant genes (ZnT1, ZnT5, ZnT7, ZIP4, ZIP5 and MTF-1) from yellow catfish Pelteobagrus fulvidraco. The six genes share similar domains to their corresponding members of mammals. Their mRNA amounts were widely existent across eight tissues (intestine, liver, brain, heart, gill, muscle, spleen and mesenteric fat), but relatively predominant in the liver and intestine. On day 28, Zn exposure tended to increase transcript levels of ZnT1, ZnT5 and MTF-1, decrease hepatic ZIP5 expression, but did not significantly affect the expression of ZnT7 and ZIP4. On day 56, Zn exposure tended to increase transcript levels of ZnT1 and MTF-1, down-regulate hepatic mRNA amounts of ZIP4 and ZIP5; among three Zn treatments, ZnT5 expression in the 0.5 mg Zn/L group and ZnT7 expression in the 0.25 mg Zn/L group were the highest. The mRNA abundances of these genes showed Zn concentration- and exposure time-dependent manners. For the first time, we characterized the full-length cDNA sequences of six Zn transport-relevant genes in fish, explored their tissue expression profiles and transcriptional responses to Zn exposure. Our study built good basis for further investigating their physiological functions of these genes and provided new insights into the regulatory mechanisms of Zn homeostasis in fish.

PPARß in yellow catfish Pelteobagrus fulvidraco: molecular characterization, tissue expression and transcriptional regulation by dietary Cu and Zn.

Peroxisome proliferator-activated receptor beta (PPARß) is a ligand-activated transcription factor that plays critical roles in the regulation of many important physiological processes. In this study, PPARß was cloned and characterized in yellow catfish Pelteobagrus fulvidraco. PPARß cDNA was 2350 bp in length with an open reading frame (ORF) of 1530 bp, encoding 509 amino acids, a 5'-untranslated region (UTR) of 474 bp, and a 3'-UTR of 346 bp. Similar to mammals, PPARß protein was predicted to consist of four domains, the A/B domain, DNA-binding domain (DBD), D domain, and ligand-binding domain (LBD). The DBD contained two zinc fingers with eight conserved cysteine residues. The predicted secondary structure of LBD consisted of 12 highly conserved α-helices and a small ß-sheet of 4 strands. In addition, PPARß was widely expressed across the tested tissues (liver, heart, muscle, intestine, brain, spleen, kidney, fat, ovary, and gill), but at the variable levels. Furthermore, the transcriptional responses of PPARß by dietary Cu and Zn levels were also investigated. Dietary Cu levels showed no significant effects on PPARß mRNA levels in the liver and intestine; in contrast, dietary Zn levels upregulated the hepatic PPARß mRNA levels, but not in the intestine. The present study serves to increase our understanding into the function of the PPARß gene in fish.

Magnesium Reduces Hepatic Lipid Accumulation in Yellow Catfish (Pelteobagrus fulvidraco) and Modulates Lipogenesis and Lipolysis via PPARA, JAK-STAT, and AMPK Pathways in Hepatocytes.

Background: Magnesium influences hepatic lipid deposition in vertebrates, but the underlying mechanism is unknown.Objective: We used yellow catfish and their isolated hepatocytes to test the hypothesis that magnesium influences lipid deposition by modulating lipogenesis and lipolysis.Methods: Juvenile yellow catfish (mean ± SEM weight: 3.43 ± 0.02 g, 3 mo old, mixed sex) were fed a 0.14- (low), 0.87- (intermediate) or 2.11- (high) g Mg/kg diet for 56 d. Primary hepatocytes were incubated for 48 h in control or MgSO4-containing medium with or without 2-h pretreatment with an inhibitor (AG490, GW6471, or Compound C). Growth performance, cell viability, triglyceride (TG) concentrations, and expression of enzymes and genes involved in lipid metabolism were measured. Results: Compared with fish fed low magnesium, those fed intermediate or high magnesium had lower hepatic lipids (18%, 22%) and 6-phosphogluconate dehydrogenase (6PGD; 3.7%, 3.8%) and malic enzyme (ME; 35%, 48%) activities and greater mRNA levels of the lipolytic genes adipose triacylglyceride lipase (atgl; 82% and 1.7-fold) and peroxisome proliferator-activated receptor (ppara; 18% and 1.0-fold), respectively (P < 0.05). Relative mRNA levels of AMP-activated protein kinase (ampk) a1, ampka2, ampkb1, ampkb2, ampkg1a, ampkg1b, Janus kinase (jak) 2a, jak2b, and signal transducers and activators of transcription (stat) 3 in fish fed high magnesium were higher (24% to 3.1-fold, P < 0.05) than in those fed low or intermediate magnesium. Compared with cells incubated with MgSO4 alone, those incubated with MgSO4 and pretreated with AG490, GW6471, or Compound C had greater TG concentrations (42%, 31%, or 56%), g6pd (98%, 59%, or 51%), 6pgd (68%, 73%, or 32%) mRNA expression, and activities of G6PD (35%, 45%, or 16%) and ME (1.5-fold, 1.3-fold, or 13%), and reduced upregulation (61%, 25%, or 45%) of the lipolytic gene, atgl (P < 0.05).Conclusions: Magnesium reduced hepatic lipid accumulation in yellow catfish and the variation might be attributed to inhibited lipogenesis and increased lipolysis. PPARA, JAK-STAT, and AMPK pathways mediated the magnesium-induced changes in lipid deposition and metabolism. These results offer new insight into magnesium nutrition in vertebrates.

Isolation and Expression Analysis of STAT Members from Synechogobius hasta and Their Roles in Leptin Affecting Lipid Metabolism.

Signal transducers and activators of transcription proteins (STATs) act as important mediators in multiple biological processes induced by a large number of cytokines. In the present study, full-length cDNA sequences of seven STAT members, including some splicing variants different from those in mammals, were obtained from Synechogobius hasta. The phylogenetic analysis revealed that the seven STAT members were derived from paralogous genes that might have arisen by whole genome duplication (WGD) events during vertebrate evolution. All of these members share similar domain structure compared with those of mammals, and were widely expressed across the tested tissues (brain, gill, heart, intestine, liver, muscle and spleen), but at variable levels. Incubation in vitro of recombinant human leptin changed the intracellular triglyceride (TG) content and mRNA levels of several STATs members, as well as expressions and activities of genes involved in lipid metabolism. Furthermore, Tyrphostin B42 (AG490), a specific inhibitor of the Janus Kinase 2(JAK2)-STAT pathway, partially reversed leptin-induced change on STAT3 and its two spliced isoforms expression, as well as expressions and activities of genes involved in lipid metabolism. As a consequence, the decrease of TG content was also reversed. Thus, our study suggests that STAT3 is the requisite for the leptin signal and the activation of the STAT3 member may account for the leptin-induced changes in lipid metabolism in S. hasta.

Effects of Fat and Fatty Acids on the Formation of Autolysosomes in the Livers from Yellow Catfish Pelteobagrus Fulvidraco.

The autophagy-lysosome pathway, which involves many crucial genes and proteins, plays crucial roles in the maintenance of intracellular homeostasis by the degradation of damaged components. At present, some of these genes and proteins have been identified but their specific functions are largely unknown. This study was performed to clone and characterize the full-length cDNA sequences of nine key autolysosome-related genes (vps11, vps16, vps18, vps33b, vps41, lamp1, mcoln1, ctsd1 and tfeb) from yellow catfish Pelteobagrus fulvidraco. The expression of these genes and the transcriptional responses to a high-fat diet and fatty acids (FAs) (palmitic acid (PA) and oleic acid (OA)) were investigated. The mRNAs of these genes could be detected in heart, liver, muscle, spleen, brain, mesenteric adipose tissue, intestine, kidney and ovary, but varied with the tissues. In the liver, the mRNA levels of the nine autolysosome-related genes were lower in fish fed a high-fat diet than those fed the control, indicating that a high-fat diet inhibited formation of autolysosomes. Palmitic acid (a saturated FA) significantly inhibited the formation of autolysosomes at 12 h, 24 h and 48 h incubation. In contrast, oleic acid (an unsaturated FA) significantly induced the formation of autolysosomes at 12 h, but inhibited them at 24 h. At 48 h, the effects of OA incubation on autolysosomes were OA concentration-dependent in primary hepatocytes of P. fulvidraco. The results of flow cytometry and laser confocal observations confirmed these results. PA and OA incubation also increased intracellular non-esterified fatty acid (NEFA) concentration at 12 h, 24 h and 48 h, and influenced mRNA levels of fatty acid binding protein (fabp) and fatty acid transport protein 4 (fatp4) which facilitate FA transport in primary hepatocytes of P. fulvidraco. The present study demonstrated the molecular characterization of the nine autolysosome-related genes and their transcriptional responses to fat and FAs in fish, which provides the basis for further exploring their regulatory mechanism in vertebrates.

Oxidative stress and mitochondrial dysfunction mediated Cd-induced hepatic lipid accumulation in zebrafish Danio rerio.

The present study was performed to determine the effect of waterborne CdCl2 exposure influencing lipid deposition and metabolism, oxidative stress and mitochondrial dysfunction, and explore the underlying molecular mechanism of cadmium (Cd)-induced disorder of hepatic lipid metabolism in fish. To this end, adult zebrafish were exposed to three waterborne CdCl2 concentrations (0(control), 5 and 25 µg Cd/l, respectively) for 30 days. Lipid accumulation, the activities of enzymes related to lipid metabolism and oxidative stress, as well as the expression level of genes involved in lipid metabolism and mitophagy were determined in the liver of zebrafish. Waterborne CdCl2 exposure increased hepatic triglyceride (TG) and Cd accumulation, the activities of fatty acid synthase (FAS), 6-phosphogluconate dehydrogenase (6PGD), glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), and the mRNA level of fatty acid synthase (fas), acetyl-CoA carboxylase alpha (acaca), glucose 6-phosphate dehydrogenase (g6pd) and malic enzyme (me), but reduced the mRNA level of carnitine palmitoyl transferase 1 (cpt1), hormone-sensitive lipase alpha (hsla), and adipose triacylglyceride lipase (atgl). The activities of superoxide dismutase (SOD), glutathoinine peroxidase (GPx) and cytochrome c oxidase (COX) and the ATP level were significantly reduced after CdCl2 exposure. CdCl2 exposure significantly increased the mRNA level of genes (microtubule-associated protein light chain 3 alpha (lc3a), PTEN-induced putative kinase 1 (pink1), NIP3-like protein X (nix) and PARKIN (parkin)) related to mitophagy. To elucidate the mechanism, reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and the mitochondrial permeability transition (MPT) inhibitor cyclosporine A (CsA) were used to verify the role of ROS and mitochondrial dysfunction in Cd-induced disorder of lipid metabolism. NAC pretreatment reversed the Cd-induced up-regulation of TG accumulation and activities of lipogenic enzymes, and the Cd-induced down-regulation of mRNA levels of lipolytic genes. Meanwhile, NAC pretreatment also blocked the mitochondrial membrane potential (MMP) collapse and decreased the ATP level, suggesting that ROS played a crucial role in regulating the Cd-induced mitochondrial dysfunction. Taken together, our findings, for the first time, highlight the importance of the oxidative stress and mitochondrial dysfunction in Cd-induced disorder of hepatic lipid metabolism, which proposed a novel mechanism for elucidating metal element exposure inducing the disorder of lipid metabolism in vertebrates.

Identification of apoptosis-related genes Bcl2 and Bax from yellow catfish Pelteobagrus fulvidraco and their transcriptional responses to waterborne and dietborne zinc exposure.

Apoptosis plays a key role in the physiology of multicellular organisms, and has been well studied in mammals, but not in teleosts. Zinc (Zn) has been shown to be an important regulator of apoptosis and apoptosis involves in the regulation of lipid metabolism. Moreover, our recent study indicated that waterborne and dietborne Zn exposure differently influenced lipid metabolism in Pelteobagrus fulvidraco, but further mechanism remained unknown. The hypothesis of the present study is that apoptosis mediated the Zn-induced changes of lipid metabolism of P. fulvidraco subjected to different exposure pathways. To this end, we cloned full-length cDNA sequences of Bcl2 and three Bax subtypes involved in apoptosis in P. fulvidraco, explored their mRNA expressions in responses to different Zn exposure pathways. Bcl2 and three Bax subtypes shared similar domain structure as typical pro- and anti-apoptotic Bcl2 family members. Their mRNAs were widely expressed among various tissues, but at variable levels. Waterborne Zn exposure down-regulated mRNA levels of Baxg and ratios of Baxa/Bcl2, and Baxg/Bcl2, but showed no significant effects on mRNA abundances of Bcl2, Baxa and Baxb, and the ratio of Baxb/Bcl2. In contrast, dietborne Zn exposure up-regulated mRNA levels of Bcl2, Baxa, Baxb and Baxg, but reduced the ratios of Baxa/Bcl2, Baxb/Bcl2, and Baxg/Bcl2. Considering their important roles of these genes in apoptosis induced by Zn, apoptosis may mediate the Zn-induced changes of hepatic lipid metabolism of Pelteobagrus fulvidraco under different Zn exposure pathways. For the first time, we characterized the full-length cDNA sequences of Bcl2 and three Bax subtypes, determined their expression profiles and transcriptional responses to different Zn exposure pathways, which would contribute to our understanding of the molecular basis of apoptosis, and also provide new insights into physiological responses to different Zn exposure pathways.

Identification of autophagy related genes LC3 and ATG4 from yellow catfish Pelteobagrus fulvidraco and their transcriptional responses to waterborne and dietborne zinc exposure.

Autophagy mediates the regulation of lipid metabolism. Moreover, our recent study indicated that waterborne and dietborne zinc (Zn) exposure differentially influenced lipid metabolism in a fish species of significance for aquaculture, yellow catfish Pelteobagrus fulvidraco, but further mechanism remained unknown. The hypothesis of the present study is that autophagy mediated the Zn-induced changes of lipid metabolism of yellow catfish subjected to different exposure pathways. To this end, we cloned key genes involved in autophagy in yellow catfish, explored their mRNA expressions in responses to different Zn exposure pathways. Full-length cDNA sequences of two LC3 subtypes and six ATG4 isoforms were isolated from yellow catfish. More ATG4 members were firstly identified in fish that might have arisen by teleost-specific whole genome duplication events. All of these members shared similar domain structure to their orthologous genes of vertebrates. Their mRNAs were widely expressed in various tissues, but at variable levels. Extra Zn addition in water or diets induced (P < 0.05) mRNA expression of ATG4Da, ATG4Db and LC3B. Considering their important roles of these genes in lipid metabolism, ATG4Da, ATG4Db and LC3B may mediate the changes of Zn-induced hepatic lipid metabolism of yellow catfish under different Zn exposure pathways. For the first time, we characterized the full-length cDNA sequences of six ATG4 isoforms and two LC3 subtypes, determined their tissue expression profiles and transcriptional responses to different Zn exposure pathways, which would contribute to our understanding of the molecular basis of autophagy, and also provide new insights into physiological responses to different Zn exposure pathways.

Identification of eight copper (Cu) uptake related genes from yellow catfish Pelteobagrus fulvidraco, and their tissue expression and transcriptional responses to dietborne Cu exposure.

The present working hypothesis is that absorption of dietary Cu is related to mRNA expressions of genes involved in Cu uptake and transport of the intestine in fish. To this end, the full-length cDNA sequences of eight Cu uptake related genes, including two isoforms of copper transporter genes (ctr1 and ctr2), three copper chaperone genes (atox1, ccs and cox17), two Cu-ATPase genes (atp7a and atp7b) and divalent metal ion transporter 1 (dmt1), were cloned and characterized in yellow catfish P. fulvidraco, respectively. Their mRNA tissue expression and transcriptional responses to dietborne Cu exposure were investigated. Compared to the corresponding members of mammals, all of these members in P. fulvidraco shared the similar conserved domain structures. Their mRNAs were expressed in a wide range of tissues (including liver, muscle, spleen, brain, gill, intestine, heart and kidney), but at variable levels. In anterior intestine, mRNA levels of ctr1, cox17, dmt1 and atp7a declined with increasing dietary Cu levels. The mRNA levels of ctr2 and mt were the highest for excess dietary Cu group and showed no significant differences between other two treatments. Atox1 mRNA levels were the highest for Cu-deficient group and showed no significant differences between other two treatments. The mRNA levels of ccs were the highest for Cu-deficient group, followed by Cu-excess group and the lowest for adequate-Cu group. In contrast, atp7b mRNA levels were the highest for Cu-excess group and the lowest for adequate Cu group. In the mid-intestine, mRNA levels of ctr1, ctr2, atox1, ccs, cox17, dmt1 and atp7a declined with increasing dietary Cu levels. Atp7b mRNA levels were the lowest for adequate Cu group and showed no significant differences between other two treatments. Mt mRNA levels were the lowest for adequate Cu group and highest for Cu-excess group. For the first time, our study cloned and characterized ctr1, ctr2, atox1, ccs, cox17, atp7a, atp7b and dmt1 genes in P. fulvidraco and determined their tissue-specific expression, and transcriptional responses in the anterior and mid-intestine of yellow catfish under dietborne Cu exposure, which shed new light on the Cu uptake system and help to understand the molecular mechanisms of Cu homeostasis in fish.

Endoplasmic reticulum (ER) stress and cAMP/PKA pathway mediated Zn-induced hepatic lipolysis.

The present study was performed to determine the effect of Zn exposure influencing endoplasmic reticulum (ER) stress, explore the underlying molecular mechanism of Zn-induced hepatic lipolysis in a fish species of significance for aquaculture, yellow catfish Pelteobagrus fulvidraco. We found that waterborne Zn exposure evoked ER stress and unfolded protein response (UPR), and activated cAMP/PKA pathway, and up-regulated hepatic lipolysis. The increase in ER stress and lipolysis were associated with activation of cAMP/PKA signaling pathway. Zn also induced an increase in intracellular Ca2+ level, which could be partially prevented by dantrolene (RyR receptor inhibitor) and 2-APB (IP3 receptor inhibitor), demonstrating that the disturbed Ca2+ homeostasis in ER contributed to ER stress and dysregulation of lipolysis. Inhibition of ER stress by PBA attenuated UPR, inhibited the activation of cAMP/PKA pathway and resulted in down-regulation of lipolysis. Inhibition of protein kinase RNA-activated-like ER kinase (PERK) by GSK2656157 and inositol-requiring enzyme (IRE) by STF-083010 differentially influenced Zn-induced changes of lipid metabolism, indicating that PERK and IRE pathways played different regulatory roles in Zn-induced lipolysis. Inhibition of PKA by H89 blocked the Zn-induced activation of cAMP/PKA pathway with a concomitant inhibition of ER stress-mediated lipolysis. Taken together, our findings highlight the importance of the ER stress-cAMP/PKA axis in Zn-induced lipolysis, which provides new insights into Zn toxicology in fish and probably in other vertebrates.

Effect of dietary choline levels on growth performance, lipid deposition and metabolism in juvenile yellow catfish Pelteobagrus fulvidraco.

The present experiment was conducted to determine the effect and mechanism of dietary choline levels on growth performance and lipid deposition of yellow catfish Pelteobagrus fulvidraco. Dietary choline was included at three levels of 239.2 (control (without extra choline addition), 1156.4 and 2273.6mg choline per kg diet, respectively) and fed to yellow catfish (mean initial weight: 3.45±0.02g mean±standard errors of mean (SEM)) for 8weeks. Fish fed the diet containing 1156.4mgkg-1 choline showed the higher weight gain (WG), specific growth rate (SGR) and feed intake (FI), but the lower feed conversion rate (FCR), than those in control and highest choline group. Hepatosomatic index (HSI) and hepatic lipid content declined with increasing dietary choline levels. Muscle lipid content was the lowest for fish fed adequate choline diets and showed no significant difference between other two groups. Choline contents in liver and muscle increased with increasing dietary choline levels. Dietary choline levels significantly influenced mRNA levels of genes involved in lipid homeostasis in muscle and liver, such as CTP:phosphocholine cytidylyltransferase a (CCTa), phosphatidylethanolamine N-methyl-transferase (PEMT), microsomal triglyceride transfer protein (MTP), apolipoprotein b (APOBb), apolipoprotein E (ApoE) and lipoprotein lipase (LPL), and effects of dietary choline levels on lipid deposition and metabolism were tissue-specific. Different responses of these genes at the mRNA levels partially explained the profiles of lipid deposition in liver and muscle for fish fed different choline diets. To our knowledge, this is the first to explore the effect of dietary choline level on mRNA expression of these genes, which provides new insights into choline nutrition in fish.